This group has characterized a novel disorder of iron metabolism aceruloplasminemia and the molecular basis of this disease in several families. In this autosomal recessive disorder, patients present in adulthood with evidence of ceruloplasmin deficiency and a marked parenchymal iron accumulation resulting in progressive organ damage and mortality. To define the mechanisms of iron accumulation in aceruloplasminemia and the role of ceruloplasmin in iron homeostasis, an animal model of aceruloplasminemia was developed by homologous recombinant targeting of the murine ceruloplasmin gene. This ceruloplasmin-null mouse (Cp-/-) has provided an essential model for the elucidation of the cellular and molecular mechanisms of iron accumulation in this disease and reveals an essential role for ceruloplasmin in regulating iron efflux. The long-term objective of these studies is to define the cellular and molecular determinants of iron metabolism. The research in this proposal is intended to elucidate the physiologic mechanisms of cellular iron efflux by generating mice deficient in multi-copper oxidases (ceruloplasmin and hephaestin (SLA)) and in iron efflux proteins (ceruloplasmin and hereditary hemochromatosis gene (HFE) product). The role of ceruloplasmin in cellular iron efflux will be further studied by primary cell culture systems void of ceruloplasmin and transgenic Cp(-/-) mice expressing murine ceruloplasmin under control of different tissue-specific promoters. Determining the mechanisms of iron efflux may permit the development of new therapeutic strategies to prevent or ameliorate iron overload in a variety of human diseases. Ultimately, the PI proposes to apply these advancements in our knowledge of iron trafficking towards the understanding of the biological roles of iron in human nutrition. Four specific aims are proposed: (1) To examine the mechanisms of ceruloplasmin-mediated cellular iron efflux by administering heat-damaged 59Fe erythrocytes and isolating primary hepatocytes and bone-marrow derived macrophages from wildtype and ceruloplasmin knockout (CP-/-) mice and by examining the kinetics of iron efflux from the cells as well as analyzing the effects of exogenously added ceruloplasmin or transferrin. ( 2) To determine the precise role of ceruloplasmin in cellular iron efflux by generating transgenic CP-/- mice that express ceruloplasmin under control of liver or macrophage-specific promoters, and measuring iron kinetics. (3) To characterize the specific role of hephaestin in cellular iron efflux by generating double knockouts deficient in ceruloplasmin and the SLA gene for hephaestin. (4) To examine the role of the hemochromatosis gene (HFE) in cellular efflux by generating double knockouts in CP and HEF mice.